The switch from latency to lytic phase of Epstein-Barr virus (EBV) is coordinated by the expression of two viral transactivators known as ZEBRA and RTA. The BFRF1 gene has been shown to be transcribed during the early phases of EBV lytic cycle. Here, we characterized the BFRF1 promoter showing that ZEBRA transfection stimulated BFRF1 expression, whereas RTA induced BFRF1 only after the transfection of an amount of plasmid largely in excess than that sufficient to stimulate the expression of other RTA-responsive genes. However, a co-operative effect between ZEBRA and RTA in the expression of BFRF1 is evident since the transfection of RTA can rescue the transactivating capacity of a mutant of the ZEBRA protein, known as Z(S186A), that has a substitution affecting the DNA binding region. Moreover, we identified one ZEBRA-responsive element (ZRE) and one RTA-responsive element (RRE) within the BFRF1 promoter region.